Biochemical Characterization Of Recombinant Woodchuck Vascular Endothelium Growth Factor (Wvegf)

Vascular endothelial growth factor (VEGF) is one of the most potent and predominant angiogeneic signals. It binds receptors on blood vessels and stimulates proliferation, migration, survival and permeability of endothelial cells that form new vasculature for malignant tumors. Human hepatocellular carcinoma (HCC) is a highly VEGF-driven tumor and frequently occurs in the context of chronic hepatitis B or C virus infection. The woodchuck is a well characterized model of hepatitis B virus related HCC as it develops acute and chronic infection after neonatal infection with sera containing woodchuck hepatitis virus. This is a valuable model for translational studies of novel VEGF targeted agents as results may have great clinical relevance. We cloned a VEGF cDNA (wVEGF) from woodchuck liver tissue and transiently expressed it in COS-1 cells. A variety of parameters were measured to functionally characterize the wVEGF. Woodchuck cell lines, namely WCH17, WHK44 (woodchuck HCC), WC3 (woodchuck hepatocyte) and HepG2 cells were also characterized. The wVEGF peptide sequence was 94% identical to the human VEGFA. Two protein bands were detected in the supernatant (secreted) with molecular weights of approximately 25 and 17kDa, and a single band of 25kDa in the cell lysates. The major band observed in the cell lines was 25kD. The amount of wVEGF protein in the supernatant was 14-fold higher than in the lysate. The mRNA level in the cell lysate was significantly higher than was present in either the COS cells transfected with empty vector and also in the woodchuck and HepG2 cell lines. After 72 hours of stimulation with supernatant wVEGF, COS cells exhibited approximately 34% higher growth compared to cells in absence of wVEGF. The antiproliferative property of sunitinib, (a VEGF receptor tyrosine kinase inhibitor) on COS cells overexpressing wVEGF showed similar IC 50 values for MTS assay 7.6±1.4 µM and for cell death (cell counts), 6.4±1.5 µM. In the cell lines, sensitivity to sunitinib ranked from WHK44 (4.5±1.2 µM) > WC3 (4.8±1.3 µM) >HepG2 (9.5±1.4 µM) >WCH17 (17.9±1.4 µM). In summary, the wVEGF was 94% identical in amino acid sequence to the human VEGF 189. It produced two protein bands that were associated with the supernatant after overexpressing the wVEGF in COS cells, one protein band was associated with the cell lysate. The wVEGF protein stimulated cell growth and exhibited sensitivity to the sunitinib with reduction in cell proliferation. (Support:American Cancer Society MRSG-08-096-01-CCE, Roswell Park Alliance Grant, PI Iyer) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5156. doi:10.1158/1538-7445.AM2011-5156